Purification of a cell growth inhibitor from bovine cerebral cortex cells

A glycopeptide, isolated from bovine cerebral cortex cells and added in only nanogram levels to cells in culture, has been shown to inhibit both cell protein synthesis and cell division. When purified by gel filtration and $\text{Ulex}$$\text{europaeus}$ lectin affinity chromatography, the radioiodinated preparation was subjected to high resolution isoelectric focusing and shown to contain three species of macromolecules. The glycopeptide focusing at pH 8.1 comprised over 75% of the radioiodinated material and possessed inhibitory activity against both cell protein synthesis and cell division. A second species that focused at pH 8.3 was also found to be inhibitory to cell metabolism and may have represented a variant of the major glycopeptide.     

Purification, properties and temperature dependence of the adenosine deaminase from a poikilotherm (bay scallop)

The adenosine deaminase of the digestive diverticulum of the bay scallop was purified and electrophoresis of the purified enzyme yielded a single enzymatically active band at several different pH values. A molecular weight of 130,000 was estimated using gel filtration and sucrose density gradient centrifugation. The enzyme had spectral properties typical of simple proteins and its isoelectric point proved to be 4.8. The scallop enzyme was stable at room temperature from pH 5.0 to 7.0, and in this range it was exceptionally resistant to heat inactivation.The effect of the substrate, adenosine, on the reaction velocity was followed over a 10,000-fold concentration range, and no deviation from Michaelis-Menten kinetics was observed. The following rate equation applies to the enzyme: $\text{1}\text{^v}\text{= (}\text{1}\text{α[}\text{S}\text{]}\text{) + (}\text{1}\text{β}\text{)}$.The effect of pH on the reaction, using adenosine as the substrate, was studied; and it was found that pH had a much greater effect on the α parameter of the rate equation than on the β parameter and that pH had little effect on the apparent activation energy of either parameter. The apparent activation energy of the β parameter was 12.2 kcal with adenosine as the substrate, while the apparent activation energy of the α parameter was zero. The α parameter of the rate equation, using other substrates, was also insensitive to temperature.     

Purification of the insecticidal toxin from the parasporal crystal of Bacillus thuringiensis subsp. kurstaki

The insecticidal toxin of $\text{Bacillus}\text{thuringiensis}$ subsp. $\text{kurstaki}$ was isolated from parasporal crystals. The toxin, which is stable for several months, is a glycoprotein with an apparent molecular weight of 68,000 that is generated upon solubilization and activation of a higher molecular weight protoxin (MW_app = 1.3 × 10⁵) at alkaline pH. The toxin was purified by gel filtation and anion exchange chromatography and its molecular weight was established by gel filtration chromatography and SDS polyacrylamide gel electrophoresis.     

Purification and evidence for heterogeneity of acid phosphatase from Saccharomycescerevisiae

The protoplast-secreted acid phosphatase of yeast $\text{Saccharomyces}\text{cerevisiae}$ was purified about 60 fold by ultrafiltration, gel filtration and chromatography on DEAE-Sephadex A-25. It was established that the enzyme is free of inactive proteins as well as polysaccharides and contains 48% of neutral sugars. The failure to separate the protein from the carbohydrates by several procedures indicates that the carbohydrate part is covalently linked to the protein. A pronounced heterogeneity of the enzyme with respect to charge as well as to molecular weight was found. The data obtained by gel filtration indicated enzyme heterogeneity in respect to carbohydrate content.     

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were $\text{N-}\text{acetylmuramic}$ acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 166 mg/l with purified Micrococcus cell walls and the V_max value was 0.56 A₅₄₀ unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly-l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.     

Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues

Four major ALDH isozymes have been identified in human tissues using starch gel electrophoresis and isoelectric focusing. The isozyme bands have been termed as ALDH I, II, III and IV according to their decreasing electrophoretic migration and increasing isoelectric point. The isozymes have been partially purified via preparative isoelectric focusing. Kinetic characteristics of ALDH I and II were found to be quite similar to ALDH enzyme 2 and enzyme 1 described earlier by Greenfield and Pietruszko (Biochem Biophys Acta, $\text{483}$ 35–45 1977). ALDH III and IV showed a very high Km for propionaldehyde (1.0–1.5 mM at pH 9.5) and were not inhibited by disulfiram at pH 9.5. A variant phenotype of ALDH which lacked in isozyme I was detected in various tissues from Japanese individuals. Comparative kinetic properties of normal and variant enzyme are given.     

Purification of a new glutathione S-transferase (transferase μ) from human liver having high activity with benzo(α)pyrene-4,5-oxide

A new glutathione $\text{S}$-transferase from human liver has been purified to homogeneity in good yield by use of ion-exchange chromatography on DEAE-cellulose, affinity chromatography on $\text{S}$-hexylglutathione coupled to epoxy-activated Sepharose 6B, and chromatography on hydroxyapatite. This new enzyme, transferase μ, is present in high concentration, but only in some individuals. It has an isoelectric point at about pH 6 to 6.5 and a different substrate specificity than the previously described alkaline transferases α-ε from human liver. Especially noteworthy is the finding of high activity against benzo(α)pyrene-4,5-oxide. Glutathione $\text{S}$-transferase μ has about 20-fold higher activity with this substrate than have the alkaline transferases. The most pronounced difference was found with $\text{trans}$-4-phenyl-3-buten-2-one which was >100-fold better as substrate for transferase μ than for the previously described transferases.     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

Glutathione S-transferases from the New Zealand grass grub,Costelytra zealandica Their isolation and characterization and the effect on their activity of endogenous factors

Isolation of glutathione $\text{S-}\text{transferase}$ from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates.Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 23,700 and the pI 5.9 enzyme one of subunit $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 22,500.A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme.     

Isolation and purification of the sexual agglutination substance of mating type a cells in Saccharomyces cerevisiae

The substance responsible for the sexual agglutinability was successfully solubilized by a newly established autoclaving method from the surface of mating type $\text{a}$ cells of $\text{Saccharomyces cerevisiae}$ and purified by DEAE cellulose chromatography, gel filtration, affinity chromatography and electrophoresis. The substance was found to consist of at least two different glycoprotein subunits. The molecular weight of the substance was estimated to be about 23,000 daltons by gel filtration. The substance was univalent in its biological activity and specifically masked the sexual agglutinability of the mating type α cells. The substance formed a complementary complex with the agglutination substance from α cells in vitro.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, $\text{O-(α-2-}\text{sulphamino}\text{-2-}\text{deoxy-}\text{D}\text{-glucopyranosyl}\text{)-(1→3)-}\text{L}\text{-[6,}{}^3\text{H]}\text{idonic}$ acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent M_r of the haloenzyme as determined by gel filtration was 190 000 ± 20 000. Two other larger M_r protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoreced as a single major band with an apparent M_r corresponding to 55 000 ± 6000.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

Purification and properties of malic enzyme from Pseudomonas diminuta IFO-13182

A malic enzyme from a cell-free extract of Pseudomonas diminuta IFO-13182 was purified to electrophoretic homogeneity by DEAE-Sepharose, Sephacryl, and Blue-Sepharose chromatographies. The purified enzyme required either NAD⁺ or NADP⁺ as a coenzyme. From the results of coenzyme specificity, the enzyme should be classified as l-malate: NAD⁺ oxidoreductase (decarboxylating) [EC 1.1.1.39]. The purified enzyme was most active at pH 7.5 and 50°C and was stable in the pH range from 7.0 to 9.0. The isoelectric point was pH 4.3. Its molecular weight was 680,000 by COSMOSIL 5-Diol high performance liquid gel filtration on chromatography and 65,000 by SDS polyacrylamide gel electrophoresis. This indicates that the enzyme consisted of 10 subunits. The malic enzyme activity with NADP⁺ was about twice that measured with NAD⁺.     

Purification and some properties of trehalase from Chaetomium aureum MS-27

The trehalase of Chaetomium aureum was purified about 196-fold with a yield of 51% from the culture filtrate by ammonium sulfate fractionation, DEAE-cellulose column chromatography, acetone fractionation, and Sephadex G-100 gel filtration. The enzyme preparation was homogeneous on disc electrophoresis. The enzyme was most active at pH 4.0 and 50°C. The enzyme was stable from pH 4.0 to 9.0 on 12 h incubation at 37°C. The molecular weight of the enzyme was estimated to be 450,000 by gel filtration on a column of Sepharose 6B, and 115,000 by SDS polyacrylamide gel electrophoresis. This indicated that the enzyme might consist of 4 subunits. The isoelectric point of the enzyme was pH 4.0. The enzyme was active specifically on trehalose and not active on the other disaccharides tested.     

Study on a proteolytic enzyme from Trypanosoma congolense

Summary A protease has been purified from Trypanosoma congolense bloodstream forms by osmotic disruption, freeze-thawing of the cells, followed by chromatography using Thiopropyl-Sepharose and gel filtration.The enzyme is a thiolprotease. A combination of SDS-polyacrylamide gel electrophoresis and contact print zymograms using casein as substrate showed a single proteolytic band with a molecular weight of 31 000. The isoelectric point of the enzyme as ascertained by isoelectric focusing extended from pH 4.4 to 5.5 with a maximum at pH 5.0. The protease cleaved various heat denatured substrates such as casein, hemoglobin, albumin and ovalbumin. The highest enzyme activity was observed at pH 5.5 and pH 6.0 using casein and hemoglobin as substrates respectively. The max. temperature was found to be 50 °C. The enzyme is inactivated by mercurial compounds, iodoacetamide, iodoacetate, chloromethylketones and leupeptin and is activated by dithioerythritol.     

Isolation of an acyl-CoA carboxylase from the tapeworm spirometra mansonoides

An acyl-CoA carboxylase, which catalyzes the carboxylation of acetylpropionyl-, and butyryl-CoA, has been isolated from the tapeworm $\text{Spirometra}\text{mansonoides}$. The enzyme has an absolute requirement for ATP, Mg²⁺, and HCO₃${}^{\mathrm{?}}$ and, in addition, requires K⁺ for full catalytic activity. The enzyme has been purified 50-fold by a combination of calcium phosphate gel adsorption, ion-exchange column chromatography, and gel filtration. In its substrate specificity, K⁺ requirement, molecular size, and antigenic behavior, the tapeworm enzyme is similar to the acyl-CoA carboxylase of another helminth— the free-living nematode $\text{Turbatrix}\text{aceti}$.     

Further purification and characterization of trehalases from the American cockroach,Periplaneta americana

Summary A soluble trehalase was purified more than 200-fold from the male accessory gland of the American cockroach,Periplaneta americana, by CM-cellulose, hydrophobic chromatography, and Sephacryl S-200 gel filtration. The final preparation was homogeneous as judged by polyacryl-amide gel electrophoresis in the absence and presence of SDS, isoelectric focusing, and immuno-diffusion tests. The purified enzyme was maximally active at pH 5.2, and showed high specificity for trehalose with aK _m of 0.98 mM. The isoelectric point was 4.7. The molecular weight of the enzyme (75,000) was determined by molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis. The amino acid composition was determined and compared with those of trehalases purified from other sources. The trehalase could be stained for carbohydrate with the periodic acid-Schiff's reagent following SDS-polyacrylamide gel electrophoresis, indicating that it was a glycoprotein. Another soluble trehalase and two types of fat body trehalases could be highly purified by the method described. A comparison of the properties of trehalases from the accessory gland and the fat body showed some resemblance.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.